Studies on asexual stage immunity to P. falciparum 1.) Evaluate the merozoite antigen PfRH5 as a vaccine candidate. Our collaborators at Oxford University (Dr. Simon Draper et al.) are pursuing this protein as a vaccine candidate and, as a prelude to that, have conducted an immunization-challenge trial in Aotus monkeys. We have collaborated on evaluating growth inhibition activity (GIA) in sera from the monkeys and these results have now been published; the promising results have supported a human clinical trial of PfRH5 which has recently been completed; we have analyzed results from this trial and a manuscript reporting on this is in preparation. In addition, we are preparing for another clinical trial of PfRH5 which will be initiated later this year. 2.) Investigate other targets of merozoite immunity - AMA1 While AMA1 is a prominent vaccine candidate, it has two issues:1) antigenic polymorphism so that antibody responses tend to be strain-specific and 2) insufficient antibody production to result in protective immunity in humans. Our data supports the use of a mixture of 4-5 different AMA1 alleles to overcome the polymorphism. Further, we have collaborated with LMVR investigators (Dr. Louis Miller) to show that using AMA1 in conjunction with its partner protein RON2 produces antibodies with greater activity in the GIA assay. This complex is required for triggering junction formation between the merozoite and the red cell surface. Evaluation of this combination in an immunization-challenge trial in a non-human primate (Aotus) has been conducted and the results have been submitted for publication by Dr. Miller, supporting a human clinical trial. In addition, we have contributed to a clinical trial of AMA1 in the UK. This trial showed that, even though high levels of antibody were elicited and those antibodies showed GIA activity, they did not affect the rate of parasite growth in the parasite-challenged vaccine recipients. Thus much more has to be done to achieve partial protection in humans with this vaccine. 3.) Studies of immunity to malaria in Kenieroba, Mali. Our 4-year investigation of the acquisition of immunity to malaria in Malian children represents perhaps the most detailed longitudinal study of malaria in African children that has been conducted and the results have now been published. More recently we collaborated with others to broaden the scope of anti-parasite functional activities and have added the neutrophil-dependent antibody-dependent respiratory burst assay. A manuscript on this technique has been published. 4.) We continue to work with Drs. Amy Bei and Dyann Wirth (Harvard University), who are studying changes in frequency of different P.falciparum clones over time in Senegal. We have worked with them to examine the influence of human immune responses on the changes in clonal parasite patterns, particularly related to the observation of parasites with common genetic signatures. The first results of these studies using GIA and the variant surface antigen assay were published and an expanded study has recently been completed and a manuscript submitted. Studies on parasite sexual stages and malaria transmission: 1.) Develop quantitative methodology for analysis of the standard mosquito membrane feeding assay (SMFA) to evaluate transmission blocking activity. The gold standard assay to evaluate the ability of antibodies to block transmission to mosquitoes is the SMFA, and we have performed an in depth study of this assay to define its characteristics in order to have confidence in assessing potential transmission blocking vaccine (TBV) candidates. We have shown that the SMFA is quite reproducible at high concentrations of antibody but highly variable at low concentrations, and we have worked with the Biostatistics group at NIAID to develop a mathematical model of the assay. Recently we have tested a modified model of the assay to show for the first time that we can predict the impact of a specific antibody on the reduction of P. falciparum parasite prevalence in mosquitoes based on the reduction in oocysts in an SMFA and the number of oocysts in control mosquitoes. Reduction in malaria prevalence in mosquitoes by an antibody is key to reducing transmission in the field. These results have recently been published and a detailed mathematical treatment of this work by our statistical colleagues is in press. 2.) Search for and evaluate new possible transmission blocking vaccine candidates. Using SMFA we have evaluated a number of potential transmission blocking vaccine candidates. In collaboration with colleagues at Oxford University we have produced and tested a number of known and unknown sexual stage proteins from P. falciparum. Sera from immunized mice have been evaluated by SMFA but none have shown high levels of oocyst inhibition. Further we have tested antibodies produced by collaborators to a number of other sexual stage and mosquito vaccine candidates to compare their activity. 3.) Assess the presence of asexual and sexual stage parasites in residents of Kenieroba, Mali throughout the year. In the spring of 2013 we initiated a new study (NIH 13-I-N107) to address the limited information on transmission in malaria endemic areas, and in 2014 we completed the field aspect of this study. Volunteers representing all age groups were finger pricked twice per month for 1 year to collect DNA and RNA. During the 2014-2016 time frame we completed analysis of over 10,000 samples of parasite DNA on filter paper. In November, 2013 (wet season), P. falciparum prevalence in the cohort was 37.0%, and in May of 2014 (dry season), the prevalence dropped to 10.0%. We also analyzed the longitudinal prevalence of the cohort; we observed that the 9-16 years old age group had the highest median longitudinal prevalence compared to the other age groups, and males had a higher median than females (22.0%). 4.)We showed for the first time that increasing P.falciparum longitudinal prevalence throughout the year was associated with decreasing risk of clinical malaria. This suggests that those with persistent parasite carriage acquire stronger protective immunity against clinical malaria. 5.) We added a new barcoding procedure this year based on a methodology developed at Harvard University to determine how many different clones of parasites an individual was carrying. This allowed us to show that at least 70% of infections were polygenomic, so that most people in the population are carrying more than one parasite clone at any given time. 6.) Using the RNA collections from the same study, we used a qRT-PCR procedure to identify Pfs25 mRNA specific for gametocytes. In 2015-2016 we completed RNA analysis to determine who is carrying gametocytes. Interestingly, even though you might not see them on microscopy, more than 80% of people with parasites also have detectable gametocytes by molecular criteria. This shows that no one group can be uniquely targeted for interventions to reduce transmission. 7.) With support from PATH/MVI, we have begun to evaluate humanized monoclonal antibodies to 3 different sexual/mosquito stage antigens. 8.) We have initiated studies to examine the differentiation pattern of P. falciparum gametocytes in culture for 3 weeks using RNASeq techniques. We have obtained data on RNA transcription during differentiation and we are analyzing the data to identify better markers of early and late stage gametocyte development. In addition, we are conducting a series of experiments to determine what factors are necessary for successful differentiation of gametocytes so that robust numbers of oocysts can be obtained after mosquito infection.